tailieunhanh - Antibody Phage Display Methods and Protocols - part 5

Pha loãng trong PBSM 2% ở nồng độ khuyến cáo của nhà cung cấp. 8. 10X Tetramethylbenzidine đệm (TMB). Hòa tan 37,4 g Na acetate-3H2O trong 230 ml H2O. Điều chỉnh độ pH bằng axit citric bão hòa (92,5 g acid citric-50 mL H2O) và điều chỉnh âm lượng đến 250 ml | 150 Chames Hoogenboom and Henderikx Fig. 2. ELISA using biotinylated antigen and soluble antibody fragments. 6. Anti-tag monoclonal Ab . 9E10 for myc-tagged Abs . Dilute in 2 PBSM according to the supplier s recommendations. 7. Rabbit anti-mouse peroxidase RAMPO . Dilute in 2 PBSM at a concentration recommended by the supplier. 8. 10X Tetramethylbenzidine buffer TMB . Dissolve g Na acetate-3H2O in 230 mL of H2O. Adjust the pH with saturated citric acid g citric acid-50 mL H2O and adjust the volume to 250 mL. 9. TMB stock. Dissolve 10 mg TMB in 1 mL DMSO. 10. TMB staining solution. Mix 1 mL 10X TMB buffer with 9 mL H2O microtiter plate. Add 100 pL TMB and 1 pL 30 hydrogen peroxidase. Make this solution fresh and keep it in the dark. 11. 96-Well flat-bottomed ELISA microtiter plates 2 plates to screen 96 colonies . 12. For IE microtiter plates with low coating efficiency 2 96 colonies . 13. Microtiter plate reader for optical density 450 nm OD450 measurements . 3. Methods . Biotinylation of Ag This method describes chemical biotinylation which is the most common way to obtain a biotinylated Ag. For other alternatives see Notes 1-3. . Chemical Biotinylation of Ag 1. Dissolve the peptide protein of interest at a concentration of 1-10 mg mL in 50 mM NaHCO3 pH . If the peptide protein is in another solvent dialyze for at least 4 h against 1 L 50 mM NaHCO3 changing the buffer 2-3x. 2. Calculate the amount of NHS-SS-Biotin required using a molar ratio of biotin protein between 5 and 20 1 see Note 5 . 3. Dissolve the required amount of NHS-SS-Biotin in dH2O see Note 6 and immediately add to the protein sample or alternatively when using larger amounts of protein add NHS-SS-Biotin directly to the protein solution. Ab Selection Against Biotinylated Ags 151 4. Incubate for 30 min at room temperature or for 2 h on ice if the protein is temperature-sensitive. 5. Add 1 M Tris-HCl pH to a final concentration of 50 mM and incubate for 1 h on ice to block